In recent years, an increasing number of formulations of pharmacologically active proteins or peptides have been developed for commercial use. However, such drugs usually have a short half-life in the blood and most of them are injections that must be administered repeatedly at frequent intervals, thus imposing excessive burdens on patients during drug administration. Hence, there is a demand for practical, sustained-release formulations of protein or peptide drugs, which exert their efficacy in as small amounts as possible and which permit reduced frequency of administration.
Sustained-release formulations of pharmacologically active proteins or peptides will cause denaturation or aggregation of the proteins or peptides during formulation preparation or sustained release, which results in a reduced recovery rate and constitutes a major obstacle to their development for commercial use. It has been attempted to prepare sustained-release formulations based on a biodegradable polymer matrix such as polylactic acid-polyglycolic acid copolymer (PLGA), but such formulations have been reported to cause protein denaturation and/or aggregation due to the hydrophobicity of the matrix, a drying step and/or a decrease in pH (see Non-patent Documents 1 and 2). On the other hand, there are also reports of sustained-release formulations based on a hydrophilic hydrogel matrix with reduced risks of these problems, but such formulations are not ready for commercial use. In terms of safety, a material used as a sustained-release matrix should combine non-antigenicity, non-mutagenicity, non-toxicity and biodegradability. Thus, no sustained-release formulation is now ready for commercial use in all aspects, i.e., encapsulation efficiency and recovery rate of proteins or peptides, as well as safety.
Some recent reports have proposed the use of polysaccharides as matrixes for drug carriers. Among them, hyaluronic acid (HA), a biomaterial (polysaccharide) isolated from the vitreous body of bovine eyes in 1934 by K. Meyer, has been known as a major component of extracellular matrix for a long time. HA is a kind of glycosaminoglycan composed of disaccharide units in which D-glucuronic acid and N-acetylglucosamine are linked to one another via β(1→3)glycosidic linkages.
There is no difference among species in the chemical and physical structure of HA and humans also have a metabolic system for HA; HA is therefore one of the safest medical biomaterials in terms of immunity and toxicity. Recent years have enabled microbial mass production of high-molecular-weight HA and also have allowed commercial use of HA in the fields of therapeutic agents for degenerated cartilage, cosmetics, etc.
There are also many reports of crosslinking techniques for HA as a matrix and sustained release formulations of protein or peptide drugs from HA gels. Techniques known for gelling HA via chemical crosslinking include the carbodiimide method (see Patent Document 1), the divinylsulfone method (see Patent Document 2), and the glycidyl ether method (see Patent Document 3). In general, when a protein or peptide is introduced into a crosslinked gel for encapsulation purposes, it results in a low introduction efficiency because of problems arising from compatibility and electrostatic repulsion between HA and the protein or peptide. In contrast, when in situ crosslinking is performed in the presence of a protein or peptide, it is advantageous in that the protein or peptide can be held in a gel at a high encapsulation efficiency. There are some reports showing that such in situ crosslinking is adapted for encapsulation of proteins or peptides into HA gels to give sustained-release formulations (see, e.g., Patent Document 4). However, there arises a problem of recovery rate when such an approach is used for in situ crosslinking of HA in the presence of proteins or peptides to prepare sustained-release formulations. As an example, a method is reported in which a HA derivative (HA-HZ) modified to have a hydrazide group (HZ) is crosslinked with a crosslinking agent comprising N-hydroxysuccinimide (NHS) (see Patent Document 5). This method is intended for in situ crosslinking under physiological conditions and limits crosslinkage formation at pH 7.4 to pH 8.5. However, the inventors' investigations have confirmed that this method also results in low recovery rates of proteins or peptides from the thus obtained HA gel. This is because the proteins or peptides will be partially reacted (mainly at their amino groups) with the crosslinking agent during crosslinking reaction to give crosslinked proteins. This method also suffers from a problem in that denatured proteins or peptides remaining in the gel have reduced biological activity and, if anything, are responsible for the cause of antigenicity. Although it is an essential requirement for pharmaceutical preparations that the encapsulated drug is released at a high recovery rate, no method is known for chemically crosslinking and gelling HA without causing proteins or peptides to react. Also, another method has been reported to encapsulate proteins or peptides at high recovery rates, in which polyethylene glycol (PEG) is used as a matrix and crosslinked through nucleophilic addition reaction of unsaturated functional groups (see Patent Document 6), but this method suffers from a problem in that fragments of non-biodegradable PEG remain unabsorbed.
In actual fact, to formulate such sustained-release materials into injectable formulations, these materials should be formulated in the form of microparticles. Spray dryers are widely used in such attempts and are also reported to be used in formulating insulin (see Non-patent Documents 3 and 4) and rh anti-IgE antibody (see Non-patent Document 5) in the form of microparticles, as well as in encapsulated drugs into hyaluronic acid microparticles (see Patent Documents 7 and 8). However, since the microparticles thus obtained will be dissolved in a short time in the subcutaneous tissues, they have a very short period of sustained release and are less practical for sustained release purposes. There is another report in which chitosan is crosslinked during spray drying so as to encapsulate low-molecular drugs therein (see Non-patent Document 6). However, the release period is as short as a few minutes in this case, and aldehyde used as a crosslinking agent is highly reactive with a functional group such as an amino group and hence cannot be used for proteins, peptides and other low-molecular drugs having a functional group(s) such as an amino group.    Patent Document 1: International Publication No. WO94/02517    Patent Document 2: JP 61-138601 A    Patent Document 3: JP 5-140201 A    Patent Document 4: U.S. Pat. No. 5,827,937    Patent Document 5: International Publication No. WO95/15168    Patent Document 6: International Publication No. WO00/44808    Patent Document 7: Japanese Patent No. 3445283    Patent Document 8: International Publication No. WO96/18647    Non-patent Document 1: J. Pharm. Sci., vol. 88, pp. 166-173, 1999    Non-patent Document 2: J. Microencapsulation, vol. 15, pp. 699-713, 1998    Non-patent Document 3: Int. J. Pharm. 233, 227-237, 2002    Non-patent Document 4: J. Control. Rel. 91, 385-394, 2003    Non-patent Document 5: Biotech. and Bioeng. 60, 301-309, 1998    Non-patent Document 6: Int. J. Pharm. 187, 53-65, 1999